Background: The Repeat Expansion Diseases are caused by intergenerational expansions of a specific tandem repeat. More than 20 such diseases have been identified thus far. Expansion of a CGGCCG-repeat in the 5' UTR of the FMR1 gene is associated with 3 different clinical presentations: Individuals with 60-200 repeats, the so-called premutation allele, are at risk for Fragile X-associated tremor-ataxia syndrome whose symptoms include in addition to neurodegeneration, loss of autonomic function including bowel and urinary incontinence. Female carriers of premutation alleles are also at risk of primary ovarian insufficiency. Furthermore, in females, the premutation allele can undergo expansion on intergenerational transfer that can result in their children having alleles with >200 repeats. This expanded allele is known as a full mutation and individuals who inherit such alleles almost always have Fragile X syndrome (FXS), which is associated with intellectual disability, autistic symptoms, digestive difficulties, and behavior problems including aggression and depression. The mechanism by which is expansion occurs is unknown. It is thought to differ from the generalized microsatellite instability seen in many different cancers in that the instability is confined to a single genetic locus and shows a strong expansion bias. Repeat expansion diseases caused by small increases in repeat number frequently show a paternal bias, while those diseases resulting from large increases in repeat number are frequently maternally derived. Whether this reflects 2 different mechanisms is unknown. Expanded alleles are also associated with a folate-sensitive fragile site that is coincident with the repeat on the X chromosome. This site, which gives the disorder its name, is one of many fragile sites present on the human genome. These sites are prone to breakage and in some cases are associated with deleterious chromosome deletions and translocations.[unreadable] [unreadable] Progress report: We have generated FXS premutation mice containing 120 CGGCCG-repeats in the 5 UTR of the endogenous murine Fmr1 gene (Entezam et al., 2007). We have shown that the repeat in these animals shows instability resembling the instability seen in human carriers of premutation alleles in its frequency and expansion bias. However most of these expansions are small and have a paternal expansion bias. This is reminiscent of what is seen in human carriers of smaller premutation alleles. [unreadable] [unreadable] We have now crossed these mice to mice with mutations in the ATR gene, a gene that encodes an important DNA damage checkpoint protein. Heterozygosity of the ATR null allele results in a large increase in the expansion frequency but only when the allele is maternally transmitted (Entezam and Usdin, 2008). No difference in repeat size is seen in various tissues of in young animals suggesting that this increased expansion is not somatic in origin. Heterozygous offspring of heterozygous mothers have a higher expansion frequency than WT offspring of the same mothers. However, even the expansion frequency seen in WT animals is higher than that seen when the mothers are WT for ATR. Thus expansion probably occurs during gametogenesis both while the gamete is diploid and when it is haploid. Expansion occurring in the haploid gamete indicates that the underlying mechanism probably does not involve replication problems arising during meiotic S-phase. It is more likely to involve some sort of aberrant DNA damage repair process. Aberrant repair of DNA damage during gametogenesis may contribute to the maternal bias in large expansions since gametogenesis lasts decades in human females compared to weeks in male mice and humans and weeks or months in female mice. Thus the window of opportunity for generating these expansions is much larger than it would be in males of both species and in female mice.[unreadable] [unreadable] We have also found evidence for somatic instability in these animals. This instability is only seen in relatively old animals and is confined to liver, brain and gonads. In some instances expansion can be so extensive in brain that nothing is left of the original allele. Since many brain cells are postmitotic, this supports the idea that these ATR-sensitive expansions are related to DNA damage repair rather than replication. Since FXTAS risk and disease severity are related to repeat number, similar expansions occurring in the brains of humans may have biological consequences. Work is currently underway to identify the source of DNA damage that gives rise to the ATR-sensitive expansions in mice and to assess somatic expansion risk in humans.[unreadable] [unreadable] We have also shown that agents that induce fragility of the Fragile X fragile site activate the ATR DNA damage response pathway and that expression of this site can be induced by knocking down or inhibiting components of this pathway. This suggests that chromosome fragility and repeat expansion may share a common mechanism.